The present invention relates to methods for determining the presence or absence of organisms of the Chlamydiaceae family in endocervical, throat and urethral swabs, urine, sputa, bronchoalveolar lavage fluids, eye secretions or other patient and animal specimens, cultures, food, and environmental samples. The method involves using nucleic acid primers to amplify specifically a target sequence within the ribonuclease P RNA gene (rnpB), preferably using one of the techniques of Strand Displacement Amplification (SDA), thermophilic Strand Displacement Amplification (tSDA) or fluorescent real time tSDA.
Three species in the family Chlamydiaceae: Chlamydophila pneumoniae (formerly Chlamydia pneumoniae), Chlamydia trachomatis and Chlamydophila psittaci (formerly Chlamydia psittaci) (Everett, et al., 1999, Int. J. Syst. Bacteriol. 49:415-440), cause diseases in humans, which include trachoma; respiratory infection (pneumonia); and sexually transmitted infections of the reproductive organs, such as urethritis, cervicitis, pelvic inflammatory disease, and epididymitis. Pneumonia can be caused by C. pneumoniae and C. psittaci (psittacosis) in adults, and by C. trachomatis in newborn infants (Guo, et al., 1995, Clin. Microbiol. Rev. 8:451-461 and Madico, et al., 2000, J. Clin. Microbiol. 38(3):1085-1093). Some species of the Chlamydiaceae family may also be involved in infections of the heart (Odeh, et al., 1992, Eur. J. Clin. Microbiol. Infect. Dis. 11:885-893). The two genera in the family Chlamydiaceae, Chlamydia and Chlamydophila, include several species that can cause diseases in animals (Everett, et al., Vet. Microbiol. 75(2):109-126). Among these are, C. psittaci and C. pecorum which give rise to a wide variety of conditions in animals including abortion, pneumonia, enteritis, polyarthretis, encephalomyelitis, and conjunctivitis (Sheehy, et al., 1996, J. Clin. Microbiol. 34(2):3175-3179). There is therefore a clinical need for the detection of all pathogens or potential pathogens within the Chlamydiaceae in a variety of clinical samples.
Endoribonuclease P (RNase P) is a ribonucleoprotein complex that removes 5xe2x80x2 leader sequences from tRNA precursors during tRNA biosynthesis. RNase P is an essential riboenzyme found in all living cells and subcellular compartments that synthesize tRNA, although catalytic proficiency of the RNA alone has been demonstrated only for the bacterial RNase P (Brown, et al., 1992, Nucl. Acids Res. 20:1451-1456 and Haas, et al., 1998, Nucl. Acids Res. 26:4093-4099). Sequencing of the RNase P RNA genes provides a potential tool for the identification of bacteria and eukaryotic organisms in clinical diagnostics. The RNase P RNA gene (rnpB) has recently been used as a marker to differentiate chlamydial strains and species (Herrmann, et al., 1996, J. Clin. Microbiol. 34(8):1897-1902). Characterization of the rnpB gene in the order Chlamydiales (Herrmann, et al., 2000, Int. J. Syst. Evol. Microbiol. 50:149-158) revealed similarities of 76.6% between C. trachomatis and C. pneumoniae, 79.5% between C. trachomatis and C. psittaci, and 84.7% between C. pneumoniae and C. psittaci. It is therefore possible to use the rnpB gene as a genus or familial marker to identify organisms within the Chlamydiaceae.
Nucleic acid amplification is a powerful technology, which allows rapid detection of specific target sequences and it is therefore a promising technology for the rapid detection and identification of species in the Chlamydiaceae family. Various nucleic acid amplification methods have been described previously for the detection of some or all of the species within this group of organisms. Touchdown enzyme time release-PCR has been used to amplify specific DNA sequences in the variable regions of the 16S and 16-23S spacer rRNA genes of C. trachomatis, C. pneumoniae, and C. psittaci (Madico, et al., 2000, J. Clin. Microbiol. 38:1085-1093). Rapid detection of the Chlamydiaceae and other families in the order Chlamydiales has also been reported using three different PCR assays targeting the ompA gene and the rRNA intergenic spacer region (Everett, et al., 1999, J. Clin. Microbiol. 37:575-580). Identification of nine species of the Chlamydiaceae using PCR-Restriction Fragment Length Polymorphism analysis was also reported by Everett, et al. (1999, Int. J. Syst. Bacteriol. 49:803-813), and Jantos, et al. described detection of C. pneumoniae using a PCR-enzyme immunoassay based on the 16S rRNA gene (1998, J. Clin. Microbiol. 36:1890-1894). Kaltenbxc3x6ck, et al. established a nested PCR for genus-specific amplification of the Chlamydia ompl locus (1997, J. Clin. Microbiol. 35:1835-1841) and the application of a nested, multiplex PCR based on the 16S rRNA gene, in the investigation of psittacosis outbreaks was reported by Messmer, et al. (1997, J. Clin. Microbiol. 35:2043-2046). Differentiation of C. psittaci and C. pecorum by species-specific PCR targeting the outer membrane protein genes has been reported by Sheehy, et al. (1996, J. Clin. Microbiol. 34:3175-3179). Use of the ligase chain reaction (LCR) has also been described for the amplification and detection of C. trachomatis (Dille, et al., 1993, J. Clin. Microbiol. 31:729-731 and Schachter, et al., 1994, J. Clin. Microbiol. 32:2540-2543). The oligonucleotide primers of the present invention are applicable to nucleic acid amplification and detection of organisms belonging to the Chlamydiaceae family.
The following terms are defined herein as follows:
An amplification primer is a primer for amplification of a target sequence by extension of the primer after hybridization to the target sequence. Amplification primers are typically about 10-75 nucleotides in length, preferably about 15-50 nucleotides in length. The total length of an amplification primer for SDA is typically about 25-50 nucleotides. The 3xe2x80x2 end of an SDA amplification primer (the target binding sequence) hybridizes at the 3xe2x80x2 end of the target sequence. The target binding sequence is about 10-25 nucleotides in length and confers hybridization specificity on the amplification primer. The SDA amplification primer further comprises a recognition site for a restriction endonuclease 5xe2x80x2 to the target binding sequence. The recognition site is for a restriction endonuclease, which will nick one strand of a DNA duplex, when the recognition site is hemimodified, as described by G. Walker, et al. (1992. Proc. Natl. Acad. Sci. USA 89:392-396 and 1992 Nucl. Acids Res. 20:1691-1696). The nucleotides 5xe2x80x2 to the restriction endonuclease recognition site (the xe2x80x9ctailxe2x80x9d) function as a polymerase repriming site when the remainder of the amplification primer is nicked and displaced during SDA. The repriming function of the tail nucleotides sustains the SDA reaction and allows synthesis of multiple amplicons from a single target molecule. The tail is typically about 10-25 nucleotides in length. Its length and sequence are generally not critical and can be routinely selected and modified. As the target binding sequence is the portion of a primer which determines its target-specificity, for amplification methods which do not require specialized sequences at the ends of the target the amplification primer generally consists essentially of only the target binding sequence. For example, amplification of a target sequence according to the invention using the Polymerase Chain Reaction (PCR) will employ amplification primers consisting of the target binding sequences of the amplification primers described herein. For amplification methods that require specialized sequences appended to the target other than the nickable restriction endonuclease recognition site and the tail of SDA (e.g., an RNA polymerase promoter for Self-Sustained Sequence Replication (3SR), Nucleic Acid Sequence-Based Amplification (NASBA) or the Transcription-Based Amplification System (TAS)), the required specialized sequence may be linked to the target binding sequence using routine methods for preparation of oligonucleotides without altering the hybridization specificity of the primer.
A bumper primer or external primer is a primer used to displace primer extension products in isothermal amplification reactions. The bumper primer anneals to a target sequence upstream of the amplification primer such that extension of the bumper primer displaces the downstream amplification primer and its extension product.
The terms target or target sequence refer to nucleic acid sequences to be amplified. These include the original nucleic acid sequence to be amplified, the complementary second strand of the original nucleic acid sequence to be amplified and either strand of a copy of the original sequence which is produced by the amplification reaction. These copies serve as amplifiable targets by virtue of the fact that they contain copies of the sequence to which the amplification primers hybridize.
Copies of the target sequence that are generated during the amplification reaction are referred to as amplification products, amplimers or amplicons.
The term extension product refers to the copy of a target sequence produced by hybridization of a primer and extension of the primer by polymerase using the target sequence as a template.
The term family-specific refers to detection, amplification or oligonucleotide hybridization to organisms of species belonging to a family without substantial detection, amplification or oligonucleotide hybridization to other organisms of species belonging to a different family.
The term assay probe refers to any oligonucleotide used to facilitate detection or identification of a nucleic acid. Detector probes, detector primers, capture probes, signal primers and reporter probes as described below are examples of assay probes.
A signal primer comprises a 3xe2x80x2 target binding sequence that hybridizes to a complementary sequence in the target and further comprises a 5xe2x80x2 tail sequence that is not complementary to the target (the adapter sequence). The adapter sequence is an indirectly detectable marker selected such that its complementary sequence will hybridize to the 3xe2x80x2 end of the reporter probe described below. The signal primer hybridizes to the target sequence at least partially downstream of the hybridization site of an amplification primer. The signal primer is extended by the polymerase in a manner similar to extension of the amplification primers. Extension of the amplification primer displaces the extension product of the signal primer in a target amplification-dependent manner, producing a single-stranded product comprising a 5xe2x80x2 adapter sequence, a downstream target binding sequence and a 3xe2x80x2 binding sequence specific for hybridization to a flanking SDA amplification primer. Hybridization and extension of this flanking amplification primer and its subsequent nicking and extension creates amplification products containing the complement of the adapter sequence which may be detected as an indication of target amplification.
A reporter probe according to the present invention functions as a detector oligonucleotide and comprises a label which is preferably at least one donor/quencher dye pair, i.e., a fluorescent donor dye and a quencher for the donor fluorophore. The label is linked to a sequence or structure in the reporter probe (the reporter moiety) which does not hybridize directly to the target sequence. The sequence of the reporter probe 3xe2x80x2 to the reporter moiety is selected to hybridize to the complement of the signal primer adapter sequence. In general, the 3xe2x80x2 end of the reporter probe does not contain sequences with any significant complementarity to the target sequence. If the amplification products containing the complement of the adapter sequence described above are present, they can then hybridize to the 3xe2x80x2 end of the reporter probe. Priming and extension from the 3xe2x80x2 end of the adapter complement sequence allows the formation of the reporter moiety complement. This formation renders the reporter moiety double-stranded, thereby allowing the label of the reporter probe to be detected and indicating the presence of or the amplification of the target.
The term amplicon refers to the product of the amplification reaction generated through the extension of either or both of a pair of amplification primers. An amplicon may contain exponentially amplified nucleic acids if both primers utilized hybridize to a target sequence. Alternatively, amplicons may be generated by linear amplification if one of the primers utilized does not hybridize to the target sequence. Thus, this term is used generically herein and does not imply the presence of exponentially amplified nucleic acids.
The present invention provides oligonucleotide primers that can be used for amplification of a target sequence found in the species of the family Chlamydiaceae. More specifically, the target sequence comprises segments of the rnpB gene. The amplification primers have been designed for high-efficiency, high-specificity amplification at elevated temperatures, such as in tSDA and the PCR, however, they are also useful in lower-temperature amplification reactions such as conventional SDA, 3SR, TAS or NASBA. An oligonucleotide reporter probe that hybridizes to the complement of target specific signal primers is used to indirectly detect the amplification products.
The oligonucleotides of the invention may be used with clinical samples from humans, such as endocervical, throat and urethral swabs, urine, sputa, bronchoalveolar lavage fluids, eye secretions or with other patient and animal specimens, cultures, food, and environmental samples, for detection and identification of nucleic acid from organisms belonging to the Chlamydiaceae family using known amplification methods. The inventive oligonucleotides and assay methods provide a means for rapidly discriminating between the species of the family Chlamydiaceae and other microorganisms, allowing the practitioner to identify these microorganisms rapidly without resorting to the more traditional procedures customarily relied upon. Since human respiratory infections caused by C. pneumoniae, neonatal pneumonia caused by C. trachomatis and psittacosis caused by C. psittaci organisms can all be treated with the same antibiotic therapy, a Chlamydiaceae family-specific assay has significant clinical value. Rapid identification of an etiological agent belonging to the Chlamydiaceae family provides information within a short period of time that can be used to determine appropriate therapeutic action.
SEQ ID NOs: 1-2 are the sequences of oligonucleotides used as upstream primers for amplification of the rnpB gene. SEQ ID NOs: 3-4 are the sequences of oligonucleotides used as downstream primers for amplification of the rnpB gene. SEQ ID NOs: 5-6 are the sequences of oligonucleotides used as upstream bumpers for SDA amplification. SEQ ID NOs: 7-8 are the sequences of oligonucleotides used as downstream bumpers for SDA amplification. SEQ ID NOs: 9-10 are the sequences of signal primers for amplification and detection of sequences within the rnpB gene. SEQ ID NO: 11 is a sequence for a reporter probe designed for detection of a sequence within the rnpB gene when used in conjunction with any of the aforementioned signal primers.